Diagnostic validation of Oxford Nanopore sequencing in genetically heterogeneous disorders | LC 25

Biography

Dr Eloisa Arbustini, certified in Cardiology, Pathological Anatomy, and Genetics, leads the Center for Inherited Diseases at the IRCCS Foundation Policlinico San Matteo in Pavia, Italy, a Medical Center of Excellence (Deliberation No. 0080/CdA 26.04.2007). She serves on the boards of scientific societies and journals and is a member of the Scientific Committee of the Istituto Superiore di Sanità under the Italian Ministry of Health.

Her expertise spans clinical, diagnostic, and research advancements in hereditary diseases.

Abstract

Short-read sequencing (SRS) has transformed clinical genetic testing, enabling detection of genetic defects in e.g. solid tumors, cardiovascular and neurological disorders, including Mendelian and mitochondrial inheritance. However, SRS has inherent limitations, including PCR-dependency, restricted read lengths that hinder the detection of deep-intronic variants, and difficulties in identifying structural variants (SVs), copy number variations (CNVs), and single nucleotide variants (SNVs)/indels. Long-read sequencing (LRS), such as that available via Oxford Nanopore Technologies devices, allows real-time sequencing of long DNA molecules. LRS offers significant advantages, including detection of deep intronic variants, rapid testing even for single cases, and improved resolution of SVs, CNVs, and SNVs/indels. We assessed the Oxford Nanopore Technologies platform's performance in detecting pathogenic and likely pathogenic (P/LP) variants identified through Illumina SRS or MLPA and confirmed via Sanger, MLPA, or qPCR. We tested and analyzed 355 samples, including 294 P/LP variant carriers and 61 controls. Panels used included 4bases HEVApro (n=111), CARDIOpro (n=183), BRaCA (n=55), and ClinExpro (n=6). Sequencing was performed on GridION and PromethION 2 devices. Data were analyzed using 4eVAR platform (4bases). Oxford Nanopore technology successfully identified all P/LP variants across panels: HEVApro detected 85 variants (70 SNV/indels and 15 CNVs), CARDIOpro identified 175 variants (152 SNV/indels and 23 CNVs), and BRaCA confirmed 28 variants (25 SNV/indels and 3 CNVs). ClinExpro identified previously undetected ENG gene CNVs, resolved DMD breakpoints, and remains under evaluation in three siblings. Our findings confirm that the diagnostic performance of Oxford Nanopore technology is comparable to SRS, with advantages in resolving complex variants. Ongoing developments are extending read lengths, enhancing diagnostic precision and clinical applications.